Deposition of beta amyloid in senile plaques and the cerebral vasculature is a well-known, but poorly understood feature of Alzheimer's disease. The recent demonstration of a neurotoxic fragment of the amyloid precursor protein suggests that amyloid may be directly involved in the pathogenesis of Alzheimer's disease (Yankner et al., 1989). This proposal involves the use of protein biochemistry, neuronal cell culture and neuropathology to elucidate the normal and pathological functions of amyloid in the nervous system. Neurotoxic amyloid proteins will be purified from the conditioned medium of cDNA-transfected cells by immunoaffinity chromatography and HPLC. Polypeptide structure-function analysis of the neurotoxic amyloid protein will involve sequencing of the purified protein(s) and the testing of synthetic amyloid peptides. The presence of amyloid neurotoxic protein in the Alzheimer's disease brain will be determined by using methods of isolation optimized on the in vitro culture system. Purified neurotoxic amyloid protein will be added to primary neuronal cultures and introduced into the rodent CNS in vivo to determine if the distribution of neuronal degeneration parallels that observed in Alzheimer's disease. The mechanism of amyloid neurotoxicity will be examined in cell culture and specific pharmacologic maneuvers to block the toxicity will be tested. The normal biological function of the amyloid precursor protein (APP) in the central nervous system is unknown. Based on preliminary observations of increased neurite outgrowth in APP-transfected cells, two forms of the amyloid precursor protein (APP) will be purified and tested for neurotrophic effects in culture and in the rodent CNS in vivo. A newly developed tissue section bioassay will be utilized to assay the effects of endogenous APP and amyloid in the rodent and human CNS. The normal patterns of APP expression will be determined in culture and in the developing rodent CNS by Western blot analysis using antibodies which can distinguish between the two forms of the APP. This multidisciplinary approach is likely to yield substantial information on the issue of amyloid biological function and has potential clinical application in helping to design therapeutic strategies for Alzheimer's disease.